Estimating Soil Labile Organic Carbon and Potential Turnover Rates Using a Sequential Fumigation–Incubation Procedure

Soil Biology & Biochemistry 37 (2005) 1923–1928 www.elsevier.com/locate/soilbio

Estimating soil labile organic carbon and potential turnover rates using a sequential fumigation–incubation procedure

X.M. Zoua,b,*, H.H. Ruanc,Y.Fua, X.D. Yanga, L.Q. Shaa

aXishuangbanna Tropical Botanical Garden, The Chinese Academy of Sciences, 88 Xuefu Road, Kunming, Yunnan 650223, China bInstitute for Tropical Ecosystem Studies, University of Puerto Rico, P.O. Box 23341, San Juan, PR 00931-3341, USA cFaculty of Forest Resources and the Environment, Nanjing Forestry University, Nanjing, China

Received 20 August 2004; received in revised form 18 January 2005; accepted 22 February 2005

Abstract

Labile carbon is the fraction of soil organic carbon with most rapid turnover times and its oxidation drives the flux of CO2 between soils and atmosphere. Available chemical and physical fractionation methods for estimating soil labile organic carbon are indirect and lack a clear biological definition. We have modified the well-established Jenkinson and Powlson’s fumigation–incubation technique to estimate soil labile organic carbon using a sequential fumigation–incubation procedure. We define soil labile organic carbon as the fraction of soil organic carbon degradable during microbial growth, assuming that labile organic carbon oxidizes according to a simple negative exponential model. We used five mineral soils and a forest Oa horizon to represent a wide range of organic carbon levels. Soil labile organic carbon varied from 0.8 mg/g in an Entisol to 17.3 mg/g in the Oa materials. Potential turnover time ranged from 24 days in an Alfisol to 102 days in an Ultisol. Soil labile organic carbon contributed from 4.8% in the Alfisol to 11.1% in the Ultisol to the total organic carbon. This new procedure is a relatively easy and simple method for obtaining indices for both the pool sizes and potential turnover rates of soil labile organic carbon and provides a new approach to studying soil organic carbon. q 2005 Elsevier Ltd. All rights reserved.

Keywords: Carbon pools; Carbon turnover rates; Labile organic carbon; Soil carbon; Soil analytical methods

1. Introduction fractionation relies on the solubility of organic carbon in oxidizer, acid or base, producing soluble organic carbon A gradually increasing flux of CO2 from soil to the (e.g. Tirol-Padre and Ladha, 2004). Although water and acid atmosphere may be accelerating global climate warming. hydrolysis (6 M HCl) extractions are recommended for Soil labile organic carbon is the most active fraction of soil identifying rapid and slow turnover carbon, respectively organic carbon with rapid turnover rates, and it changes (Paul et al., 1997), these data fail to identify the chemical substantially after disturbance and management (Coleman constituents and their biological properties (Duxbury et al., et al., 1996; Harrison et al., 1993). Parton et al. (1987) 1989). Physical fractionation methods separate soil organic defined soil labile carbon as the fraction of soil organic carbon into components as heavy and light carbon (Sollins carbon with a turnover time of less than a few years as et al., 1983; Tisdall, 1996) or as coarse and fine particulate compared to recalcitrant carbon with a turnover time of organic carbon (Cambardella and Elliott, 1992, 1993). several thousand years. Soil labile organic carbon has been However, these physical fractionation methods do not represented operationally by specific fractions derived using produce consistent results because data vary with the chemical and physical separation techniques. Chemical mineral composition and density found in different soil types, with the size and density that differ among plant * Corresponding author. Address: Institute for Tropical Ecosystem materials, and with soil aggregate consistency that fluctuates Studies, University of Puerto Rico, P.O. Box 23341, San Juan, PR with dispersibility and particle bounding properties (Sollins 00931-3341, USA. Tel.: C1 787 764 0000x2868; fax: C1 787 772 1481. et al., 1999). Estimates of light and coarse particulate E-mail address: [email protected] (X.M. Zou). organic carbon can only give qualitative information about 0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. labile organic carbon. Furthermore, estimates of organic doi:10.1016/j.soilbio.2005.02.028 carbon pools derived from the existing chemical or physical 1924 X.M. Zou et al. / Soil Biology & Biochemistry 37 (2005) 1923–1928 fractionation methods do not provide information on their China. One Inceptisol was from an alluvial deposit by a potential turnover rates, unless one uses tracers such as 14C stream bank in tabonuco forest (Zou et al., 1995) that was (Jenkinson et al., 1987; Christensen and Christensen, 1995) dominated by Dacryodes excelsa Vahl at El Verde Field or provides estimates of microbial growth rates (Harris and Station in the Luquillo National Forest (108200N, 658490W), Paul, 1994). Puerto Rico. An Entisol was obtained from landslide scars Jenkinson and Powlson (1976) developed a fumigation– within the tabonuco forest where parent materials were incubation procedure to estimate soil microbial carbon. This exposed. An Oxisol was a well-weathered tropical soil within method was later modified into a fumigation–extraction the tabonuco forest from upland areas. These three soils were technique (Sparling and West, 1988; Voroney et al., 1993). located at an elevation of about 450 m above the sea level, Jenkinson et al. (1987) further developed a model that where the monthly temperature ranges between 20.8 and predicts soil carbon fractions and their turnover time using 14.4 8C with an annual average precipitation of 3467 mm 14C generated data from long-term experiments at (Soil Survey Staff, 1995). An Ultisol was obtained from the Rothamsted. However, these microbial and model estimates cloud forest of the Luquillo Mountains at an elevation of have not provided direct measurements on the pool sizes about 950 m above the sea level. The cloud forest was and potential turnover rates of soil labile organic carbon. dominated by Tabebuia rigida (Walker et al., 1996). Annual Although soil labile organic carbon is constituted of mean temperature ranged from 17 to 20 8C with the annual amino acids, simple carbohydrates, a fraction of microbial precipitation reaching 4200 mm (Brown et al., 1983). An biomass, and other simple organic compounds, a clear Alfisol and its Oa layer were obtained from the Ailaoshan chemical or physical definition of soil labile organic carbon Nature Reserve of the Yunnan province, southwestern China is difficult if not impossible. We here present a biological (248300N, 1018000W). The subtropical evergreen-broad- definition of soil labile organic carbon as microbial leaved forest was dominated by Castanopsis watii, degradable carbon associated with microbial growth. This Lithocarpus chingdongensis, Lithocarpus xylocarpus, and biological definition includes two aspects: soil labile Machilus veridis (Young et al., 1992) with annual mean organic carbon is both chemically degradable and physi- temperature of 10.8 8C and annual mean precipitation of cally accessible by soil microbes. Organic carbon that is 1900 mm (Liu, 1993). chemically degradable but physically inaccessible by Each soil was sampled to a depth of 100 mm with five microbes due to clay mineral protection is not regarded soil cores which were bulked into a composite sample. We here as soil labile organic carbon. did not sieve the soil, but plant roots O2 mm in diameter, The objective of this study is to develop a new method for rocks, and soil macrofauna were removed. The floor mass estimating indices of biologically-defined soil labile carbon Oa layer was obtained by passing floor mass through a 3 mm pools and their potential turnover rates. We adopted Jenkinson sieve. Soils were thoroughly homogenized by hand and Powlson’s fumigation–incubation approach for estimat- kneading before analyses. Soil total carbon and N were ing soil labile carbon as the accumulated CO2 released from determined by combustion in a LECO 2000 CHN elemental microbial growth using a sequential fumigation–incubation analyzer (LECO Corporation, St Joseph, MI, USA). procedure. We assume that soil labile carbon is degraded Element concentrations of P, K, Ca, Mg, Fe, and Al were according to the simple negative exponential equation as being obtained using a Beckman DCP spectraspan V (Beckman widely seen in plant litter decomposition (Olson, 1963). The Instruments, Fullerton, CA, USA) after samples had been accumulated microbial carbon from the sequential fumi- digested with H2O2 and concentrated HNO3 (Parkinson and gation–incubation can then be estimated using a simple first- Allen, 1975). order kinetics model (Stanford and Smith, 1972). Relying on the established fumigation–incubation procedure which is 2.2. Microbial biomass and labile organic carbon relatively simple in operation, this method provides indices of both labile organic carbon pools and their potential turnover We used the modified Jenkinson and Powlson’s fumi- rates. One can obtain indices of labile organic carbon pools gation–incubation method (Liu and Zou, 2002)for and potential turnover rates within a period of less than estimating microbial biomass. A subsample of 10 g of soil 3months. was oven-dried at 105 8C for 24 h for determining soil moisture. Two subsamples of 30 g of soil (wet weight) each were used for the fumigation and control treatments. Soil 2. Materials and methods microbial biomass was calculated from difference in CO2 evolved during the first 10 days’ incubation from the 2.1. Soils fumigated and control soils. Microbial biomass was calculated as (Jenkinson and Powlson, 1976): BZF/K, We used five mineral soils and an Oa forest-floor material where B is soil biomass carbon in mg C/g soil; F is CO2–C representing a wide range of carbon levels. These soils were evolved during the 10 days’ incubation from the fumigated obtained from tropical forests in Puerto Rico and a soil, less that evolved from control soil incubated for the subtropical evergreen-broad-leaved forest in Yunnan of same time under the same conditions; and KZ0.45, or X.M. Zou et al. / Soil Biology & Biochemistry 37 (2005) 1923–1928 1925

Table 1 Chemical properties of soils used for the sequential fumigation–incubation study

Soil order C (%; GSE) N (%; GSE) P (mg/g) K (mg/g) Ca (mg/g) Mg (mg/g) Fe (mg/g) Al (mg/g) (GSE) (GSE) (GSE) (GSE) (GSE) (GSE) Entisol 1.27 (0.60) 0.08 (0.04) 0.71 (0.21) 1.33 (0.14) 0.14 (0.05) 0.74 (0.22) 73.38 (4.84) 47.67 (6.89) Inceptisol 2.71 (0.25) 0.23 (0.03) 0.29 (0.02) 1.28 (0.05) 2.00 (0.23) 6.64 (1.07) 72.33 (4.23) 42.92 (2.66) Alﬁsol 11.23 (0.61) 0.63 (0.05) 0.81 (0.04) 9.99 (0.71) 0.62 (0.05) 3.12 (0.16) 42.99 (1.59) 55.22 (3.65) Ultisol 11.87 (2.70) 0.52 (0.10) 0.15 (0.05) 1.10 (0.21) 0.37 (0.11) 0.44 (0.04) 63.10 (13.25) 31.57 (5.53) Oxisol 4.98 (0.35) 0.41 (0.02) 0.26 (0.01) 1.09 (0.11) 0.60 (0.23) 2.52 (0.72) 74.42 (2.23) 52.06 (5.90) Oaa 34.24 0.81 0.91 3.01 4.10 1.21 11.80 11.79

a Note: Data from Liu et al. (1995).

the fraction of the biomass carbon mineralization to CO2–C Entisol and highest value of 11.9% in the Ultisol from the following the fumigation–incubation. To estimate soil labile cloud forest (Table 1). Because of the inclusion of a fraction organic carbon, the fumigated subsamples were subjected to of mineral soil, the Oa materials had a total organic carbon the fumigation–incubation procedure for a total of 5–8 cycles concentration of 34.2%. Soil total nitrogen concentrations (n, the number of fumigation–incubation cycles). Carbon were the lowest in the Entisol with a value of 0.08% and dioxide evolved from the first and the subsequent incubations highest in the Alfisol with a value reaching 0.63%. of the fumigated soils was used for estimating labile organic However, the concentrations of the divalent cations Ca carbon, after adjusting for the addition of 1 g inoculation soil. and Mg were highest in the river alluvial Inceptisol. The evolved soil CO2 carbon (Ct in milligrams per gram of Concentrations of P and K were higher in the Alfisol than soil) following the tth fumigation–incubation cycle was in other soils. The Oxisol had the highest values for Fe and calculated using the modified formula of Carter (1993): the Alfisol the highest value for Al, indicating high levels of Z K K Ct [(At Vt)NtE Qt]/w, where At is the volume (milli- acidity. liters) of acid needed to titrate the NaOH in the beakers for the Soil microbial biomass carbon levels were highest in the blank; Vt is the volume (milliliters) of acid needed to titrate Alfisol (2.5 mg/g) and its Oa (4.2 mg/g) materials from the the NaOH in the beakers containing the fumigated soils; Nt evergreen-broad-leaved forest, and lowest in the landslide Z is the normality of the acid; E 6, the equivalent weight; and Entisol with a value of 0.20 mg/g of soil (Table 2). w is the initial weight of soil for fumigation treatment. Microbial biomass carbon in the Oxisol was 1.36 mg/g of Z 0 C C C Z . Qt C /(r 1) S[CtK1/(r 1)], t 1, , n, is a correction soil, similar to the 1.30 mg/g (Ruan et al., 2004)or factor for the inoculated soil after each fumigation, where 1.50 mg/g (Liu and Zou, 2002) measured previously in the Z Z 0 CtK1 0 when t 1; C is the amount of CO2–C from the same forest, but higher than the 0.7 mg/g from the adjacent control soil during the first 10-day incubation; and r is pine plantation soil (Li et al., in press). Correlations the weight ratio of fumigated soil to inoculation soil. The between Ln-transformed values of CO –C evolved from Z . 2 accumulated CO2–C (Mt, t 1, ,n) from the fumigated soil the fumigated samples and the sequential fumigation– is calculated according to Stanford and Smith (1972) incubation cycles (t) were all linear (Eq. (2)) and Z K Kkt significant (Fig. 1). Estimates of soil labile organic carbon Mt Clabileð1 e Þ; (1) pools ranged from 0.80 mg/g in the landslide Entisol where Clabile is the estimated pool size of soil labile organic to 13.17 mg/g in the Ultisol from the cloud forest with a carbon and k is the potential turnover rate. Potential turnover 165-fold difference. Labile organic carbon reached time (days) of soil labile organic carbon is calculated as the 17.27 mg/g in the Oa layer. In contrast, the potential inverse of k multiplied by 10 because each incubation period turnover rates (k) of soil labile organic carbon were fastest is 10 days in length. Values for Clabile and k can be estimated in the Alfisol and slowest in the Ultisol with the potential using linear regression with the following transformed turnover time of 24 and 102 days, respectively. equation: LnðC Þ Z LnðkC Þ Kkt; ðt Z 1; 2; .; nÞ; (2) Table 2 t labile Pool sizes and potential turnover rates (k) of soil labile organic C in various where k is the slope, Ln (kClabile) is the intercept (a), and soils as estimated by the sequential fumigation–incubation procedure a Z -1 Clabile e /k. Soil order CMicrobial CLabile k (cycles ) 10/k CMicrobial/ CLabile/ (mg/g) (mg/g) (day) CLabile (%) CTotal (%)

Entisol 0.20 0.80 0.278 36.0 25.25 6.27 Inceptisol 0.73 1.89 0.256 39.1 38.55 6.99 3. Results Alﬁsol 2.51 5.39 0.420 23.8 46.49 4.80 Ultisol 1.52 13.15 0.098 102.2 11.57 11.07 Oxisol Soil total organic carbon varied across soil types with the 1.37 3.82 0.188 53.2 35.81 7.67 Oa 4.21 17.27 0.308 32.5 24.35 5.04 lowest carbon content of 1.3% occurring in the landslide 1926 X.M. Zou et al. / Soil Biology & Biochemistry 37 (2005) 1923–1928

2 4. Discussion

1 Available information on soil labile organic carbon is scarce and inconsistent because of the lacking of a clear Ultisol 0 biological deﬁnition of labile organic carbon and available Oxisol analytical methods. Microbial carbon contributes 1–3% to Inceptisol )

t soil total organic carbon and was suggested as an index for -1 Entisol soil labile organic carbon (Paul and Clark, 1996). Our Alfisol Ln(C -2 Oa estimates of soil microbial carbon from five tropical soils all fall within this range. Although microbial biomass carbon -3 levels were similar between the Ultisol and Oxisol soils, the Oxisol (2.7%) had a much higher percentage of microbial carbon to total organic carbon than the Ultisol (1.3%). In -4 00468 contrast, the percentage of the estimated labile organic Sequential fumigation-incubation cycles carbon to soil total organic carbon reached 11.1% in the Ultisol, much higher than 7.6% in the Oxisol. These Fig. 1. Linear correlation between Ln-transformed values of CO2–C (mg/g) released from the corresponding sequential fumigation–incubation treat- contrasting data suggested a poor positive correlation ment cycles for five tropical soils and a floor mass Oa layer. The slopes and between soil microbial biomass carbon and labile organic intercepts in these correlations were used to estimate soil labile organic carbon. carbon pools and their potential turnover rates. We assumed that the decay of soil labile organic carbon follows an empirical simple negative exponential model as is widely accepted in plant litter decomposition (Olson, The accumulated CO –C evolved from the sequential 2 1963). Our data showed a well fit to this model, suggesting fumigation–incubation fitted well with Eq. (1) when plotted that there was no severe violation of this assumption. An against the sequential fumigation–incubation treatment inherent assumption that this new method to estimate labile cycles (Fig. 2). Eight round of fumigation–incubation organic carbon had in common with Jenkinson and treatments released over 50% of the estimated soil labile Powlson’s fumigation–incubation procedure was that the organic carbon pools for all soils, reaching the highest value fumigation treatment did not change the labile nature of soil of 90% in the Entisol. The fraction of soil microbial biomass organic carbon (Smith et al., 1995; Horwath et al., 1996). carbon to the labile organic carbon pool was highest in the Our data indicated that young soils such as Inceptisols Alfisol and lowest in the Ultisol with values of 46.5 and and Entisols had lower levels of microbial biomass carbon 11.6%, respectively. Labile organic carbon contributed the and labile organic carbon than older soils such as Ultisols largest proportion to total soil organic carbon for the Ultisol and Oxisols. However, these young soils did not differ soil, reaching 11.1%. This proportion varied between 4.8 from the older soils in the proportion of microbial biomass and 7.7% for other soil samples. carbon to total organic carbon. Our data also indicated that organic carbon levels in young soils were more labile than those in older soils with higher potential turnover rates and 16 shorter potential turnover time for the young soils, suggesting that older soils either contain high proportion 14 Oa of old organic materials or have high levels of organo- 12 mineral association. We introduced the concepts of 10 potential turnover rate and potential turnover time because -C (mg/g)

2 our measurements were derived from microbial growth 8 conditions altered from those in the ﬁeld. Microbial growth Ultisol can be limited by soil available pore space, carbon and 6 nutrient availability, or predation activities under ﬁeld Alfisol 4 conditions. Fumigation eliminates these constraints, creat-

Accumulated CO Oxisol ing a relatively ideal condition for microbial growth. 2 Inceptisol Because the sequential fumigation–incubation procedure Entisol 0 does not remove nutrients from soil and eliminates 02468 predation, the subsequent microbial growth is largely Sequential fumigation-incubation cycles limited by carbon availability both in total quantity and its relative lability or quality as reflected by the potential Fig. 2. Accumulated CO –C (M , mg/g) released from the sequential 2 t turnover rate k. The introduction of potential turnover rate fumigation–incubation cycles (t) as fitted to the equation MtZClabile Kkt (1Ke ), where Clabile is the estimated pool size of soil labile organic for describing labile organic carbon can be proven to be an carbon, and k is the potential turnover rate. important parameter in the future studies of carbon cycling X.M. Zou et al. / Soil Biology & Biochemistry 37 (2005) 1923–1928 1927 associated with natural and human disturbance and global Uehara, G. (Eds.), Dynamics of Soil Organic Matter in climate changes. Tropical Ecosystems. University of Hawaii Press, Honolulu, HI, This new method also provides a means for the USA, pp. 33–67. Harris, D., Paul, E.A., 1994. Measurement of microbial growth rates in soil. determination of soil recalcitrant organic carbon or slow Applied Soil Ecology 1, 227–290. turnover organic carbon as the difference between total Harrison, K.G., Broecker, W.S., Bonani, G., 1993. The effect of changing organic carbon and labile organic carbon. Unlike labile land-use on soil radiocarbon. Science 262, 725–726. organic carbon, this method does not provide information Horwath, W.R., Paul, E.A., Harris, D., Norton, J., Jagger, L., Horton, K.A., on the quality of recalcitrant organic carbon due to the 1996. Defining a realistic control for the chloroform-fumigation incubation method using microscopic counting and 14C-substrates. inability to obtain its potential turnover rates. One may use Canadian Journal of Soil Science 76, 459–467. isotopic labeled materials to obtain this information. This Jenkinson, D.S., Powlson, D.S., 1976. The effects of biocidal treatment on sequential fumigation–incubation procedure is based on the metabolism in soil-V. A method for measuring soil biomass. Soil widely established Jenkinson and Powlson’s fumigation– Biology & Biochemistry 8, 209–213. incubation technique. It is inexpensive, relatively simple in Jenkinson, D.S., Hart, P.B.S., Rayner, J.H., Parry, L.C., 1987. Modelling operation, and provides indices on both pool sizes of labile the turnover of organic matter in long-term experiments at Rothamsted. INTECOL Bulletin 15, 1–8. organic carbon and their potential turnover rates Li, Y., Xu, M., Zou, X.M., Xia, Y., in press. 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